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HIV/SIV exploits DCs in mucosal infection: mechanisms of transmission and modulation by semen associated factors

Periodic Reporting for period 1 - DCmucoHIV (HIV/SIV exploits DCs in mucosal infection: mechanisms of transmission and modulation by semen associated factors)

Reporting period: 2015-07-01 to 2017-06-30

The main aim of this project was to unravel the mechanisms associated with colorectal transmission of HIV/SIV by intestinal mononuclear phagocytes (MNP), in particular dendritic cells (DCs) and macrophages (Mf), and their modulation by semen. MNP residing in the colorectal mucosa are among the first target cells of the virus however, little to nothing is known about resident DC and Mf in the lower intestinal tract, the extent to which different subsets exist, and their role in HIV acquisition. Moreover, the distinction between macrophages and DCs in the gut is not obvious based on surface markers. This confusion rises because besides classical Mf, in the gut there is an atypical population of Mf that expresses CD11c, a marker previously associated to DCs. Among the cells that express CD11c, four different subsets are found in the mouse having different origin and function. Not much is known on what are the human and macaque counterpart gut MNP.
Therefore, taking advantage of the possibility to obtain human and Cynomolgus macaque intestinal tissues and using a multidisciplinary approach, the following studies were performed:
1) Characterization of DCs and Mf subsets in human and non-human primate colorectal tissues.
MNP have been isolated by collagenase digestion and analyzed by multicolor flow cytometry. Among the CD45+lineage-HLA-DR+ cells, Mf were distinguished from DCs on the base of the expression of the CD64 receptor. Thus, DCs were defined as CD11c+CD64- cells, whereas Mf were CD64+ and further classified in 2 subsets, which either expressed or not the CD11c (Figure 1). CD11c+CD64- DC were CD14-CD16- in the macaque colon and CD14- in the human colon, and in both species 3 populations of myeloid DC were identified: the CD103+CX3CR1- tolerogenic DC, the CD103-CX3CR1- non tolerogenic DCs and a small subset CD103-CX3CR1+ cells. Both Mf populations were CD103-CX3CR1+, and as already reported for murine cells the CX3CR1 is expressed at higher levels on Mf than on DCs. About 50% of the total Mfs expressed the CD163, an M2 specific marker, which however was mostly expressed on the CD14+ cells. Conversely, in the human colon, the CD64+CD11c+ Mf were mostly CD14- cells, whereas the CD64+CD11c- Mf were mostly CD14+. Cells were further characterized for the expression of the CD172a and CD11b, and HIV coreceptors (Figure 2 and data not shown). The distribution of the different cell subsets among the mucosa was analyzed by confocal microscopy. All the described subsets of DCs and Mf were identified. At steady state several CD103+ cells were present at subepithelial level and in between the epithelium of the lumen and of the cripts, however those cells were mostly CD3+ T cells. CD11c+, CD64+ and CX3CR1+ cells are diffused though out the mucosa but with a prevalence at the baso-medial level of the lamina propria.
2) Analysis of MNP redistribution following HIV/SIV stimulation
Ex vivo colorectal tissues were exposed in a polarized manner to R5 HIV-1 stimulation and analyzed by immunofluorescence and confocal microscopy in order to study cell subsets redistribution. Both CD11c+CD64+ and CD11c+CX3CR1+ cells penetrated the intestinal epithelium as soon as 30 minutes after virus incubation, whereas an increase in CD11c+CD103+ cells migration was not observed. Interestingly, CCR5, that we showed to drive CD11c+ cells migration toward the intestinal lumen, was preferentially expressed by the CD11c+CD64+CX3CR1+ cells, which support their involvement in active sampling of HIV and in transmission of infection in situ. Interestingly, seminal plasma from HIV+ subjects induced the same migratory properties as R5 HIV-1, highlighting the physiological role of MNP recruitment as a mechanism of virus spreading.
A possible role of CD11c+ cells (either DCs and Mf) recruitment in the intestinal epithelium in the pathogenesis of the infection was further supported by ours in vitro studies using strains of SIV which were either pathogenic and not pathogenic. Only pathogenic strains of SIV were able to induce migration of monocyte-derived DCs across an intestinal epithelial monolayer to capture virions and transfer them to receptive cells.

3) Modulation of SIV infection and MNP redistribution by seminal plasma
Mucosal exposure to infected semen accounts for the majority of HIV-1 transmission events worldwide, with rectal intercourse being the route with the highest estimated risk of transmission. While it is well known that exposure to semen induce a transient inflammation of the female reproductive tract (FRT), the effect of rectal exposure to semen in vivo and the implications for HIV transmission have been limited studied. Moreover, the effect of leukocytospermia, which is indicative of an inflammatory status of the sperm donor, has never been addressed. Using Cynomolgus macaque tissue explants from the sigmoid colon, we developed a model of mucosal exposure to seminal plasma (SP) to study SIVmac251 transmission. Explants were treated with the virus in presence and absence of SP collected from leukocytospermic and non leukocytospermic SIV- macaques. Semen from leukocytospermic individuals had significantly higher concentrations of inflammatory cytokines, including IL6, IL8, MIP1b, RANTES, and MCP1. Leukocytospermic SP enhanced virus replication as evaluated by SIV RNA viral copies in explant culture medium over time, as well as by SIV DNA quantification in explants after 2 weeks of infection (Figure 3). While no change in the tightness and integrity of the epithelium was observed, SP treatment induced recruitment of leukocytes and intraepithelial accumulation of antigen presenting cells, as evidenced by both confocal microscopy on fixed tissues and ex vivo dynamic imaging of intestinal CD45+ and HLADR+ cells using time lapse confocal microscopy. Thus, the local inflammatory response and leukocytes recruitment induced by SP exposure might favor SIV transmission at the intestinal site.
We deeply characterized the populations of intestinal MNP of both human subjects and Cynmolgus macaques, revealing a previously underestimated complexity in the phenotype and function of the intestinal MNP in human and NHP. Analysis of the redistribution of the different subsets of MNP between the site of transmission and the draining lymph nodes following in vivo challenge of Cynomolgus macaques is ongoing and will possibly reveal the relative contribution of different subsets of DC and Mf in the early events of HIV transmission at mucosal sites. Moreover, our experimental system may contribute to improve the efficacy of currently available prevention measures by understanding the mechanisms regulating immunity at the level of the colorectal mucosa, and leading to the identification of new determinants of HIV-1 transmission therein.